Variability in the efficacy of a standardized antenatal steroid treatment is not due to maternal or fetal plasma drug levels. Evidence from a sheep model of pregnancy.

Background Antenatal steroids (ANS) are standard of care for women judged to be at imminent risk of preterm delivery. Worldwide, there is significant variation in ANS dosing strategy, selection for treatment criteria, and agent choice. This, combined with very limited optimization of ANS use per se means that treatment efficacy is highly variable and the rate of respiratory distress syndrome is decreased perhaps as little as 40%. In some cases, ANS use is associated with limited benefit and potential harm. Objective We hypothesized that individual differences in maternal and fetal steroid exposure would contribute to observed variability in ANS treatment efficacy. Using a chronically catheterized sheep model of pregnancy, we aimed to explore the relationship between materno-fetal steroid exposure and ANS treatment efficacy as determined by functional lung maturation in preterm lambs undergoing ventilation. Methods Ewes carrying a single fetus had surgery to catheterize a fetal and maternal jugular vein at 119 days’ gestation. Animals recovered for 24h before being randomized to either: i) a single maternal intramuscular injection (IM) of 2ml saline (Negative Control Group, n=10); or ii) a single maternal IM of 0.25mg/kg betamethasone phosphate + acetate (ANS Group, n=20). Serial maternal and fetal plasma samples were collected from each animal over 48h before fetuses were delivered and ventilated for 30 minutes. Total and free plasma betamethasone concentration was measured by mass spectrometry. Fetal lung tissue was collected for analysis using quantitative polymerase chain reaction. Results One animal of the Control Group and one animal from the ANS Group had did not complete their treatment protocol and were removed from analyses. Animals in the ANS Group were divided into a Responder (n=12/19) Sub-Group and a Non-Responder Sub-Group (n=7/19) using a cut-off of a PaCO2 at 30 minutes ventilation within 2SD of the mean value from saline-treated Negative Control Group animals. While ANS improved fetal lung maturation in the undivided ANS group, and in the Responder Sub-Group both physiologically (blood gas and ventilation related data) and biochemically (mRNA expression related to fetal lung maturation), these values were not improved relative to saline-treated Control Group animals in the ANS Non-Responder Sub-Group. Interestingly, no differences in betamethasone distribution, clearance, or protein binding were identified between the ANS Responder and Non-Responder Sub-Groups. Conclusion This study correlated individual materno-fetal steroid exposures with preterm lung maturation as determined by pulmonary ventilation. Herein, approximately 40% of preterm lambs exposed to antenatal steroids had lung maturation not significantly different to saline-treated Control Group animals. These non-responsive animals received maternal and fetal betamethasone exposures identical to animals that had a significant improvement in functional lung maturation. These data suggest that the efficacy of ANS therapy is not solely determined by materno-fetal drug levels, and that individual fetal or maternal factors may play a role in determining treatment outcomes in response to glucocorticoid-driven signaling

Direct administration of the non-competitive interleukin-1 receptor antagonist rytvela transiently reduced intrauterine inflammation in an extremely preterm sheep model of chorioamnionitis

Background Intraamniotic inflammation is associated with up to 40% of preterm births, most notably in deliveries occurring prior to 32 weeks’ gestation. Despite this, there are few treatment options allowing the prevention of preterm birth and associated fetal injury. Recent studies have shown that the small, non-competitive allosteric interleukin (IL)-1 receptor inhibitor, rytvela, may be of use in resolving inflammation associated with preterm birth (PTB) and fetal injury. We aimed to use an extremely preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela in response to established intra-amniotic (IA) lipopolysaccharide (LPS) exposure. We hypothesized that rytvela would reduce LPS-induced IA inflammation in amniotic fluid (AF) and fetal tissues. Methods Sheep with a single fetus at 95 days gestation (estimated fetal weight 1.0 kg) had surgery to place fetal jugular and IA catheters. Animals were recovered for 48 hours before being randomized to either: i) IA administration of 2 ml saline 24 hours before 2 ml IA and 2 ml fetal intravenous (IV) administration of saline (Saline Group, n = 7); ii) IA administration of 10 mg LPS in 2 ml saline 24 hours before 2 ml IA and 2 ml fetal IV saline (LPS Group, n = 10); 3) IA administration of 10 mg LPS in 2 ml saline 24 hours before 0.3 mg/fetal kg IA and 1 mg/fetal kg fetal IV rytvela in 2 ml saline, respectively (LPS + rytvela Group, n = 7). Serial AF samples were collected for 120 h. Inflammatory responses were characterized by quantitative polymerase chain reaction (qPCR), histology, fluorescent immunohistochemistry, enzyme-linked inmmunosorbent assay (ELISA), fluorescent western blotting and blood chemistry analysis. Results LPS-treated animals had endotoxin and AF monocyte chemoattractant protein (MCP)-1 concentrations that were significantly higher at 24 hours (immediately prior to rytvela administration) relative to values from Saline Group animals. Following rytvela administration, the average MCP-1 concentrations in the AF were significantly lower in the LPS + rytvela Group relative to in the LPS Group. In delivery samples, the expression of IL-1β in fetal skin was significantly lower in the LPS + rytvela Group compared to the LPS Group. Conclusion A single dose of rytvela was associated with partial, modest inhibition in the expression of a panel of cytokines/chemokines in fetal tissues undergoing an active inflammatory response

Successful use of an artificial placenta-based life support system to treat extremely preterm ovine fetuses compromised by intrauterine inflammation

[Introduction] Ex-vivo uterine environment (EVE) therapy is an experimental intensive care strategy for extremely preterm infants born between 21 and 24 weeks of gestation. Gas exchange is performed by membranous oxygenators connected by catheters to the umbilical vessels. The fetus is submerged in a bath of synthetic amniotic fluid. The lungs remain fluid-filled and pulmonary respiration does not occur. Intrauterine inflammation is strongly associated with extremely early preterm birth and fetal injury. Presently, there are no data of which we are aware to show that artificial placenta-based systems can be used to support extremely preterm fetuses compromised by exposure to intrauterine inflammation. [Objectives] To evaluate the ability of our EVE therapy platform to support extremely preterm ovine fetuses (95 d gestational age; approximately equivalent to 24 weeks of human gestation) exposed to intrauterine inflammation for a period of 120 hours, the following primary endpoints were chosen: i) maintenance of key physiological variables within normal ranges; ii) absence of infection and inflammation; iii) absence of brain injury; and iv) gross fetal growth and cardiovascular function matching that of age-matched in utero controls. [Study Design] Ten ewes with singleton pregnancies were each given a single intraamniotic injection of 10 mg E.coli lipopolysaccharides (LPS) under ultrasound guidance 48h before undergoing surgical delivery for adaptation to EVE therapy at 95d gestation (term=150d). Fetuses were adapted to EVE therapy and maintained for 120h with constant monitoring of key vital parameters (EVE Group) before being euthanised at 100d equivalent gestational age. Umbilical artery blood samples were regularly collected to assess blood gas data, differential counts, biochemical parameters, inflammatory markers and microbial load to exclude infection. Ultrasound was conducted at 48 h after intraamniotic LPS (before surgery) to confirm fetal viability and at the conclusion of the experiments (before euthanasia) to evaluate cardiac function. Brain injury was evaluated by gross anatomical and histopathological investigations. Eight singleton pregnant control animals were similarly exposed to intraamniotic LPS at 93d gestation and were euthanized at 100 d gestation to allow comparative post-mortem analyses (Control Group). Bio-banked samples from age-matched saline-treated animals served as an additional comparison group. Successful instillation of LPS into the amniotic fluid exposure was confirmed by amniotic fluid analysis at the time of administration, and by analyzing cytokine levels in fetal plasma and amniotic fluid. Data were tested for mean differences with ANOVA. [Results] Six out of eight LPS Control Group (75%) and eight out of ten EVE Group fetuses (80%) successfully completed their protocols. Six of eight EVE Group fetuses required dexamethasone phosphate treatment to manage profound refractory hypotension. Weight and crown rump length was reduced in EVE Group fetuses at euthanasia compared to LPS Control Group fetuses (p0.05), plasma tumour necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) concentrations (p>0.05), or liver function tests between groups. Daily blood cultures were negative for aerobic and anaerobic growth in all EVE Group animals. No cases of intraventricular haemorrhage were observed. White matter injury was identified in three of six LPS Control Group fetuses and three of eight EVE Group fetuses. [Conclusions] We report the use of an artificial placenta-based system to support extremely preterm lambs compromised by exposure to intrauterine inflammation. Our data highlight key challenges (refractory hypotension, growth restriction and white matter injury) to be overcome in the development and use of artificial placenta technology for extremely early preterm infants. As such challenges appear largely absent from studies based on healthy pregnancies, additional experiments of this nature using clinically relevant model systems are essential for further development of this technology and its eventual clinical application